Novel peptide

ABSTRACT

A novel peptide and a composition including the peptide are disclosed. The composition may be a pharmaceutical composition, a quasi-drug composition, a dietary supplement, or a food composition. A polynucleotide encoding the peptide and an expression vector including the polynucleotide are also disclosed. The peptide promotes regeneration of hard tissue and is useful for treating dentin-dental pulp diseases or periodontal diseases.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a novel peptide, and more particularly,to a peptide for promoting regeneration of hard tissue and/or dentalpulp tissues and treating dentin-dental pulp diseases, and/orperiodontal diseases, a polynucleotide encoding the peptide, anexpression vector comprising the polynucleotide, and a pharmaceuticalcomposition for preventing or treating dentin-dental pulp diseasesand/or periodontal diseases comprising the peptide, a quasi-drugcomposition for preventing or alleviating dentin-dental pulp diseasesand/or periodontal diseases comprising the peptide, and a healthfunctional food composition for preventing or alleviating dentin-dentalpulp diseases and/or periodontal diseases including the peptide.

2. Description of the Related Art

Dental pulp is a richly innervated and vascularized soft connectivetissue that occupies the pulp chamber inside a tooth and extends to theouter surface of the dentin. Disorders occurring in the dental pulp arecalled dental pulp diseases.

There are many causes of dental pulp diseases, but in most cases, dentalpulp diseases are caused by a bacterial infection due to dental caries,or infections in the dental pulp through the perforation, fracture,cracks, or periodontal pocket. External wounds, abrasion, tooth cracks,or friction or heat from dental equipment may also cause dental pulpdiseases. The pulpitis caused by bacterial infection may lead to rootapex and periodontal diseases. Dental pulp diseases successivelyprogress to pulp hyperemia, pulpitis, and pulp necrosis. Pulp necrosismay lead to periapical diseases or disorders to the entire tooth becausethe death of the dental pulp prevents the blood supply to the dentalpulp and thus the entire pulp tissue is lost.

For treatment of the pulp or periapical diseases, pulp capping materialsand pulp canal filling materials are used, and calcium hydroxides, MTA(Mineral Trioxide Aggregate), Gutta-percha, etc., has been generallyused. MTA shows therapeutic effects because it has a leakage sealingability and biocompatibility. However, the use of MTA is hampered due toits relatively high cost as a dental repair material and discoloration,leading to an esthetic problem. Gutta-percha is relatively low cost andhas excellent flow characteristics. However, it is not a physiologicallyacceptable method which causes a loss of viability of the pulp. Up tonow, conservative treatments for dentin and pulp diseases have problemsof the weak or brittle teeth or reinfection.

Periodontal tissue (periodontium) is a complex organ composed ofepithelial tissue, soft connective tissue, and calcified connectivetissue. The structure of the periodontium is composed of the gingiva,periodontal ligament (PDL), cementum, and alveolar bone. Gingivalfibroblast and periodontal ligament fibroblast are major cellularcomponents of gingival soft connective tissue, and forming andmaintaining an extracellular matrix. Additionally, the gingivalfibroblast is mainly involved in maintaining the gingival connectivetissue, while the periodontal ligament fibroblast has a unique functionto form the periodontal ligament and is involved in the restoration andregeneration of adjacent alveolar bone and cementum in vivo. Whenperiodontal disease occurs, clinically, the gingival bleeding andswelling, periodontal pocket formation, and destruction of the alveolarbone may result in dental loss.

Since the ultimate goal of treating periodontal disease is to restoredamaged connective tissue, cementum and alveolar bone, for this purpose,not only regeneration of the periodontal ligament supporting thealveolar bone but also the regeneration of the alveolar bone and thecementum that can be attached by the periodontal ligament is needed.

Therefore, many studies have been actively conducted to developtherapeutic agents capable of effectively treating dentin-dental pulpdiseases or periodontal diseases. For example, Korean Patent PublicationNo. 2012-0089547 discloses a composition for forming hard tissue orregenerating dentin or pulp tissues, including ameloblasts, apical budcells, or cultures thereof as an active ingredient, and Korean PatentPublication No. 2009-0033643 discloses novel tooth stem cells derivedfrom tooth follicles and a method of culturing the same. Furthermore,Korean Patent Publication No. 2016-0105627 discloses a pharmaceuticalcomposition for treating periodontal disease comprising pre-ameloblastconditioned medium.

The present inventors had made many efforts to develop an agent capableof more effectively treating dentin-dental pulp diseases and/orperiodontal diseases, causing damage to alveolar bone and cementum. As aresult, they developed a peptide showing effects of treating a cell forpromoting regeneration of hard tissue, including dentin, bone andcementum and/or dental pulp tissue, and treating dentin-dental pulpdiseases and/or periodontal diseases, thereby completed the presentinvention.

SUMMARY OF THE INVENTION

Embodiments of the present inventive concepts may provide a peptide forpromoting regeneration of hard tissue and/or dental pulp tissues andtreating dentin-dental pulp diseases and/or periodontal diseases.

Embodiments of the present inventive concepts may also provide apolynucleotide encoding the peptide.

Embodiments of the present inventive concepts may also provide anexpression vector, including the polynucleotide.

Embodiments of the present inventive concepts may also provide apharmaceutical composition for preventing or treating dentin-dental pulpdiseases and/or periodontal diseases, including the peptide.

Embodiments of the present inventive concepts may also provide aquasi-drug composition for preventing or alleviating dentin-dental pulpdiseases and/or periodontal diseases, including the peptide.

Embodiments of the present inventive concepts may also provide a healthfunctional food composition for preventing or alleviating dentin-dentalpulp diseases and/or periodontal diseases, including the peptide.

Embodiments of the present inventive concepts may also provide a methodof preventing or treating dentin-dental pulp diseases and/or periodontaldiseases, the method including administering the composition includingthe peptide to a subject, excluding humans.

Embodiments of the present inventive concepts may also provide a methodof promoting regeneration of hard tissue including dentin, bone, andcementum and/or dental pulp tissue, the method including administeringthe composition including the peptide to a subject. Embodiments of thepresent inventive concepts may also provide the use of a peptide forpromoting regeneration of hard tissue and/or pulp tissue.

Embodiments of the present inventive concepts may also provide the useof a peptide in preventing or treating dentin-dental pulp diseasesand/or periodontal diseases.

Embodiments of the present inventive concepts may also provide a peptidecomprising an amino acid sequence of the following Formula 1:

K-Y-R1-R2-R3-R4-R5   (Formula 1)

wherein R1 and R2 are arginine(R), lysine(K), glutamine(Q) orasparagine(N), respectively;

R3, R4, and R5 are arginine(R) or lysine(K), respectively.

Embodiments of the present inventive concepts may also provide a peptidewherein R1 is glutamine(Q), R2 is arginine(R).

Embodiments of the present inventive concepts may also provide a peptidewherein R1 is arginine(R) or lysine(K), R2 is glutamine(Q).

Embodiments of the present inventive concepts may also provide a peptidewherein R1 and R2 are glutamine (Q), respectively.

Embodiments of the present inventive concepts may also provide a peptidewherein R1 is arginine (R) or lysine (K), and R2 is arginine (R).

Embodiments of the present inventive concepts may also provide a peptidewherein R1 is glutamine (Q), arginine (R) or lysine (K), and R2 islysine (K).

Embodiments of the present inventive concepts may also provide a peptidewherein R1 is arginine (R), lysine (K), glutamine (Q) or asparagine(N),R2 is arginine (R), lysine (K), glutamine (Q) or asparagine (N), atleast one of R1 or R2 is asparagine(N).

Embodiments of the present inventive concepts may also provide a peptidewherein the peptide is subjected to N- or C-terminal acetylation,amidation, or methylation; D-amino acid introduction; peptide bondmodification including CH₂—NH, CH₂—S, CH₂—S=0, CH₂—CH₂; backbonemodification; or side-chain modification.

Effect of the Invention

A peptide of the present invention exhibits excellent effects ofpromoting regeneration of hard tissue and/or pulp tissues. Therefore, itmay be widely applied to the development of a variety of agents forpreventing or treating dentin-dental pulp diseases or for preventing ortreating periodontal diseases causing damage to bone and/or cementum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the results of comparing the expressionlevels of the dentin sialophosphoprotein (Dspp), odontoblastdifferentiation marker gene, in human dental pulp cells (hDPCs) treatedwith the novel peptide of the present invention.

FIG. 1B is another graph showing the result of comparing the expressionlevels of Dspp, odontoblast differentiation marker gene, in human dentalpulp cells (hDPCs) treated with the novel peptide of the presentinvention.

FIG. 1C is a graph showing the results of comparing the expressionlevels of Nestin, odontoblast differentiation marker genes, in humandental pulp cells (hDPCs) treated with the peptide of the presentinvention.

FIG. 2A is a graph showing the results of comparing the expressionlevels of the BSP, a bone and cementum differentiation marker gene, inhuman bone marrow-derived mesenchymal stem cells (hBMSCs) treated withthe novel peptide of the present invention.

FIG. 2B is another graph showing the results of comparing the expressionlevels of the BSP, a bone and cementum differentiation marker gene, inhuman bone marrow-derived mesenchymal stem cells (hBMSCs) treated withthe novel peptide of the present invention.

FIG. 3 shows the results of measuring the amount of newly formed hardtissue using human dental pulp cells (hDPCs) for 6 weeks in vivo.

FIG. 4 shows microscopic images showing the histomorphological analysisof the hard tissue formed using human dental pulp cells (hDPCs) for 6weeks in vivo, in which A to D show the results of transplantation of acontrol implant prepared by mixing hDPCs and 100 mg HA/TCP in a 0.5%fibrin gel for 6 weeks in a mouse with a compromised immune system, andin which E to H show the results of transplantation of implant preparedby mixing hDPCs and 100 mg HA/TCP, 10 μg peptide (group 3) in a 0.5%fibrin gel for 6 weeks in a mouse with a compromised immune system,respectively (scale bar: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50μm).

FIG. 5 shows microscopic images showing the level of collagen formationin hard tissue formed using human dental pulp cells (hDPCs) for 6 weeksin vivo, in which A to D show the results of transplantation of acontrol implant prepared by mixing hDPCs and 100 mg HA/TCP in a 0.5%fibrin gel for 6 weeks in a mouse with a compromised immune system, andin which E to H show the results of transplantation of implant preparedby mixing hDPCs, 100 mg HA/TCP, and 10 μg peptide (group 3) in a 0.5%fibrin gel for 6 weeks in a mouse with a compromised immune system,respectively (scale bar: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50μm).

FIG. 6. shows immunostaining images showing the analysis of theexpression level of DSP, odontoblast differentiation marker gene, usingimmunostaining method, in hard tissue formed using human dental pulpcells (hDPCs) for 6 weeks in vivo, in which A shows the results oftransplanting the implant prepared by mixing hDPCs and 100 mg HA/TCP ina 0.5% fibrin gel in a mouse with a compromised immune system for 6weeks, in which B shows the results of transplanting the implantprepared by mixing hDPCs 100 mg HA/TCP, and 10 μg peptide (group 3) in a0.5% fibrin gel in a mouse with a compromised immune system for 6 weeks.A and B are immunostained of the formed hard tissue using anti-DSPantibody. C is a negative control of immunohistochemical analysistreated only with secondary antibodies. Arrows in A and B indicate theexpression of DSP in newly formed calcified tissue. The scale bar is 50μm.

FIG. 7. shows the result of measuring the amount of newly formed hardtissue using human dental pulp cells (hDPCs) for 12 weeks in vivo.

FIG. 8 shows microscopic images showing the histomorphological analysisof the hard tissue formed using human dental pulp cells (hDPCs) for 12weeks in vivo, in which A to D show the results of transplantation of acontrol implant prepared by mixing hDPCs and 100 mg HA/TCP in a 0.5%fibrin gel for 12 weeks in a mouse with a compromised immune system, andin which E to H show the results of transplantation of implant preparedby mixing hDPCs and 100 mg HA/TCP, 10 μg peptide (group 3) in a 0.5%fibrin gel for 12 weeks in a mouse with a compromised immune system,respectively (scale bar: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50μm).

FIG. 9 shows microscopic images showing the level of collagen formationin hard tissue formed using human dental pulp cells (hDPCs) for 12 weeksin vivo, in which A to D show the results of transplantation of acontrol implant prepared by mixing hDPCs and 100 mg HA/TCP in a 0.5%fibrin gel for 12 weeks in a mouse with a compromised immune system, andin which E to H show the results of transplantation of implant preparedby mixing hDPCs, 100 mg HA/TCP, and 10 μg peptide (group 3) in a 0.5%fibrin gel for 12 weeks in a mouse with a compromised immune system,respectively (scale bar: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50μm).

FIG. 10. shows immunostaining images showing the analysis of theexpression level of DSP, odontoblast differentiation marker gene, usingimmunostaining method, in hard tissue formed using human dental pulpcells (hDPCs) for 12 weeks in vivo, in which A shows the results oftransplanting the implant prepared by mixing hDPCs and 100 mg HA/TCP ina 0.5% fibrin gel in a mouse with a compromised immune system for 12weeks, in which B shows the results of transplanting the implantprepared by mixing hDPCs 100 mg HA/TCP, and 10 μg peptide (group 3) in a0.5% fibrin gel in a mouse with a compromised immune system for 12weeks. A and B are immunostained of the formed hard tissue usinganti-DSP antibody. C is a negative control of immunohistochemicalanalysis treated only with secondary antibodies. Arrows in A and Bindicate the expression of DSP in newly formed calcified tissue. Thescale bar is 50 μm.

FIG. 11. shows scanning electron microscope (SEM) images showing theanalysis of hard tissue formed using human dental pulp cells (hDPCs) for12 weeks in vivo, in which A shows the results of transplanting theimplant prepared by mixing hDPCs and 100 mg HA/TCP in a 0.5% fibrin gelin a mouse with a compromised immune system for 12 weeks, in which B andC show the results of transplanting the implant prepared by mixinghDPCs, 100 mg HA/TCP, and 10 μg peptide (group 3) in a 0.5% fibrin gelin a mouse with a compromised immune system for 12 weeks.

FIG. 12. shows SEM images that the dentinal tubules of the damageddentin are regenerated and closed with physiological dentin.Individually, B, C, and D of FIG. 12 are enlarged images of A of FIG.12, respectively. And, F, G, and H of FIG. 12 are enlarged images of Eof FIG. 12, respectively. (Scale bar A: 1 mm, B: 50 μm, C: 20 μm, D: 10μm, E: 1 mm, F: 50 μm, G: 20 μm, H: 10 μm)

FIG. 13. shows SEM images that the dentinal tubules exposed to thesurface of the damaged dentin is closed by physiologicalremineralization. Individually, B, C, and D of FIG. 13 are enlargedimages of A of FIG. 13, respectively. And, F, G, and H of FIG. 13 areenlarged images of E of FIG. 13, respectively. (Scale bar A: 1 mm, B:50μm, C: 20μm, D: 10μm, E: 1 mm, F: 50μm, G: 20 μm, H: 10 μm)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors conducted many studies to develop an agent capableof more effectively treating dentin-dental pulp diseases and/orperiodontal diseases. As a result, they developed a novel peptidecomprising or consisting of 7 amino acids.

The newly developed peptide was prepared by substitution of a part of anamino acid sequence of a peptide, which may exhibit a therapeutic effecton dentin or dental pulp diseases. It was confirmed that the newlydeveloped peptide might increase expression levels of Dspp (Dentinsialophosphoprotein) and Nestin which are odontoblast differentiationmarker genes, thereby showing an effect of promoting dentinregeneration, and it is moreover possible to increase the expressionlevel of the BSP (Bone sialoprotein), which is a differentiation markergene of osteoblasts and cementoblasts, thereby exhibiting an effect ofpromoting regeneration of bone and cementum.

Further, an implant including the peptide together with human dentalpulp cells was prepared, and the prepared implant was transplanted intoa subcutaneous tissue of an immunocompromised mouse, and after 6 weeksto 12 weeks, the transplanted tissue was analyzed. As a result, it wasfound that a dentin/pulp-like tissue having the most similar morphologyto a dentin/dental pulp tissue in vivo was formed, bone-like tissuehaving the most similar morphology to a bone tissue in vivo was formed.A production level of collagen was increased, and the expression levelof DSP, which is an odontoblast-specific differentiation marker, wasincreased.

Furthermore, the morphology of the transplanted tissue was examinedunder a scanning electron microscope. As a result, odontoblast-likecells along the formed hard tissue were observed, was confirmed that thedendritic cell processes also extend toward the formed hard tissue.Moreover, a typical characteristic was confirmed that osteoblast and/orcementoblast with a cubic cell-attached on the surface of the formedhard tissue.

Therefore, it can be seen that the peptide of the present invention mayexhibit effects of promoting regeneration of hard tissue and/or dentalpulp and treating dentin-dental pulp diseases and/or periodontaldiseases. The peptide of the present invention having these effects hasnever been reported so far, and the present inventors first developedit.

In an aspect, the present invention provides a peptide for promotingregeneration of hard tissue and/or dental pulp and treatingdentin-dental pulp diseases and/or periodontal diseases, the peptideincluding an amino acid sequence of the following Formula 1:

K-Y-R1-R2-R3-R4-R5   (Formula 1)

wherein R1, and R2 are arginine(R), lysine(K), glutamine(Q) orasparagine(N);

R3, R4, and R5 are arginine(R) or lysine(K), respectively.

The term “hard tissue”, as used herein, refers to a relatively hardskeletal tissue including bone, hyaline cartilage, and fibrouscartilage. In one aspect, according to the present invention, the hardtissue may include dentin, bone, and cementum.

The term “dentin”, as used herein, is also called dentine, and refers toa yellowish-white hard tissue that makes up most of a tooth. Dentin isnot exposed to the surface of the tooth, because it is covered by enamelin the tooth crown and cementum in the root. However, dentin exposuremay occur at the apical end, or the occlusal surface of the tooth crownas the enamel wears with aging. The dentin is a kind of bone-liketissue, but it is distinguished from a general bone tissue in that thecell bodies of the dentin stay in the dental pulp while their processesextend into the dentinal tubules.

The term “cementum” of the present invention refers to a thin film of aform in which the bones covering the tooth roots (root roots) and otherparts of a mammal are slightly deformed. The cementum is composed of 50%inorganic and 50% moisture-organic, yellow in color, and exhibits lowerhardness than dentin or enamel. The cementum includes periodontalligament fibers that fix the teeth to the alveolar bone, and whenbacteria are infected with the gums, the degeneration of the cementumsurrounding the teeth occurs, and the deformed cementum periodontalligament fibers that connect the teeth and the alveolar bones do notstick to the teeth and the teeth will be shaken. In order to treat suchdegeneration of cementum, a method is used to remove the degeneratedcementum and promote the formation of new cementum.

The peptide of the present invention is characterized in that it mayincrease expression levels of Dspp, and Nestin genes which areodontoblast differentiation marker genes, the expression level of theBSP (Bone sialoprotein) genes which are differentiation marker genes ofosteoblasts and cementoblasts, and when the peptide is transplantedtogether with human dental pulp cells, the human dental pulp cells forma dentin/dental pulp-like tissue and bone-like tissue.

The peptide of the present invention includes peptide variants thereofhaving a sequence including one or more amino acid residues differentfrom those of the amino acid sequence of the peptide of the presentinvention, as long as it may promote regeneration of hard tissue likedentin, bone and cementum and/or dental pulp and exhibit a therapeuticeffect on dentin-dental pulp diseases and/or periodontal diseases.

Amino acid exchanges in proteins and polypeptides, which do notgenerally alter the molecular activity, are known in the art. The mostcommonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, in bothdirections. The peptide may include peptides that have improvedstructural stability against heat, pH, etc., or improved ability topromote regeneration of hard tissue like dentin, bone, and cementumand/or dental pulp due to alteration or modification of the amino acidsequence.

Amino acid variations are made based on the relative similarity of aminoacid side-chain substituents, such as hydrophobicity, hydrophilicity,charge, size, and the like. Since all seven amino acids comprising thepeptide of the present invention correspond to hydrophilic amino acids,the relative similarity of amino acid side chain substituents is high.Accordingly, even if the amino acids comprising the peptide of SEQ IDNO: 1 are substituted with various amino acids having hydrophilicproperties, the effect of the peptide provided by the present inventioncan be exhibited as it is due to its structural similarity. For example,although glutamine which is an acidic amino acid at position 3 of thepeptide of SEQ ID NO: 1 of the present invention is substituted with anacidic amino acid, asparagine, or a basic amino acid, lysine orarginine, the effects of the peptide of the present invention may beobtained as it is; although arginine which is a basic amino acid atposition 4 of the peptide of SEQ ID NO: 1 is substituted with a basicamino acid, lysine or an acidic amino acid, glutamine or asparagine, theeffects of the peptide of the present invention may be obtained as itis; although arginine which is a basic amino acid at position 5 of thepeptide of SEQ ID NO: 1 is substituted with a basic amino acid lysine,the effects of the peptide of the present invention may be obtained asit is; although lysine which is a basic amino acid at position 6, or 7ofthe peptide of SEQ ID NO: 1 is substituted with a basic amino acidarginine, the effects of the peptide of the present invention may beobtained as it is.

As such, although the acidic amino acids or basic amino acids comprisingthe peptide of the present invention are substituted with amino acidshaving the same properties, or substituted with different acidic aminoacids or basic amino acids, respectively, the effects of the peptide ofthe present invention may be obtained as it is. Therefore, it isapparent that a peptide variant having a sequence including one or moreamino acid residues different from those of the amino acid sequenceconstituting the peptide of the present invention is also included inthe scope of the peptide of the present invention.

Further, although arbitrary amino acids are added at the N-terminus orC-terminus of the peptide of the prevention, the effects of the peptideof the present invention may be obtained as it is. Therefore, a peptideprepared by adding arbitrary amino acids at the N-terminus or C-terminusof the peptide of the present invention is also included in the scope ofthe peptide of the present invention. For example, a peptide prepared byadding 1 to 300 amino acids at the N-terminus or C-terminus of thepeptide of the present invention may be exemplified, for anotherexample, a peptide prepared by adding 1 to 100 amino acids at theN-terminus or C-terminus of the peptide of the present invention may beexemplified, and for still another example, a peptide prepared by adding1 to 24 amino acids at the N-terminus or C-terminus of the peptide ofthe present invention may be exemplified.

The peptide of the present invention may be chemically modified orprotected with an organic group at the N-terminus and/or C-terminus, ormay be modified by adding amino acids at the peptide terminus in orderto protect the peptide from protease in vivo and to increase stabilitythereof. In particular, since chemically synthesized peptides havecharged N-terminus and C-terminus, N-terminal acetylation, N-terminalmethylation, or/and C-terminal amidation may be performed, or D-aminoacid introduction, peptide bond modification such as CH₂—NH, CH₂—S,CH₂-S=0, CH₂— CH₂, backbone modification, or side-chain modification maybe included in order to remove the charge, but is not limited thereto.Methods of preparing peptidomimetic compounds are well known in the art,for example, referring to a description in Quantitative Drug Design,C.A. Ramsden Gd., Choplin Pergamon Press (1992).

The term “backbone modification”, as used herein, refers to directmodification of amino acids constituting a peptide backbone with aminoacid analogs, in which the backbone (main chain) refers to a main chain-or ring-shaped framework of amino acids constituting a peptide. Theamino acid analog refers to an amino acid modified by substitution ofhydrogen atoms on the nitrogen or a-carbon of the amino acid backbone.

The term “side-chain modification”, as used herein, refers to themodification of side-chains of amino acids by using a chemical material,in which the side-chains of amino acids refer to atomic groups branchedfrom a main chain- or ring-shaped framework of amino acids constitutinga peptide. Examples of the peptide side-chain modification may includeamino group modification such as reductive alkylation; amidation withmethyl acetimidate; alkylation with acetic anhydride; carbamylation ofamino groups with cyanate; trinitrobenzylation of amino acids with2,4,6-trinitrobenzene sulfonic acid (TNBS); alkylation of amino groupswith succinic anhydride; and pyridoxylation with pyridoxal-5-phosphatefollowed by reduction with NaBH4.

Further, the peptide of the present invention may be used alone or incombination with various carriers approved as a drug, such as an organicsolvent. In order to improve stability and efficacy, the peptide of thepresent invention may also be used by including carbohydrates such asglucose, sucrose, or dextran, antioxidants such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins, otherstabilizers, etc.

According to an embodiment of the present invention, 128 kinds ofpeptides corresponding to Formula 1 of the present invention weresynthesized, and effects of the synthesized peptides on an expressionlevel of Dspp gene, which is an odontoblast differentiation marker genewere examined. As a result, it was confirmed that all mRNA levels of theodontoblast differentiation marker Dspp gene in human dental pulp cellswhich were treated with 128 kinds of the peptides were 8 times higher, 6times higher, 3 times higher, or at least 1.5 times higher than an mRNAlevel of the Dspp gene which was measured in human dental pulp cells(control group) which were treated with none of the peptides of thepresent invention (FIG. 1A, FIG. 1B and Tables 18 to 33).

As reported up to now, it is known that as the mRNA level of DSPP isincreased, odontoblast differentiation and dentin regeneration arepromoted, and therefore, it can be seen that 128 kinds of the peptidesshowing the effect of increasing the mRNA level of Dspp gen may exhibitthe effect of promoting odontoblast differentiation and dentinregeneration (Taduru Sreenath et al., THE JOURNAL OF BIOLOGICALCHEMISTRY, Vol. 278, No. 27, Issue of July 4, pp. 24874-24880, 2003;William T.

Butler et al., Connective Tissue Research, 44(Suppl. 1): 171-178, 2003).

Further, an embodiment of the present invention, the effects of thesynthesized peptides on an expression level of BSP gene, which is adifferentiation marker gene of osteoblasts/cementoblasts, were examined.As a result, it was confirmed that all mRNA levels of the BSP,osteoblasts/cementoblasts differentiation marker BSP gene in humandental pulp cells which were treated with 128 kinds of the peptides were13 times higher, 12 times higher, 9 times higher, or at least 3 timeshigher than an mRNA level of the BSP gene which was measured in humandental pulp cells (control group) which were treated with none of thepeptides of the present invention (FIG. 2A, FIG. 2B).

It is known that as the mRNA level of BSP is increased,osteoblasts/cementoblasts differentiation and bone and cementumdifferentiations are promoted, and therefore, it can be seen that 128kinds of the peptides showing the effect of increasing the mRNA level ofBSP gene may exhibit the effect of promoting osteoblasts/cementoblastsand odontoblast differentiation and dentin regeneration. In anotheraspect, the present invention provides a polynucleotide encoding thepeptide.

The polynucleotide may be modified by substitution, deletion, orinsertion of one or more bases, or a combination thereof. When thenucleotide sequence is prepared by chemical synthesis, a syntheticmethod widely known in the art, for example, a method described in aliterature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988)may be used, and the nucleotide sequence may be synthesized by triester,phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on solid supports, etc. Forexample, the polynucleotide encoding the peptide of the presentinvention may include a nucleotide sequence of SEQ ID NO: 4.

In still another aspect, the present invention provides an expressionvector including the polynucleotide, a transformant including theexpression vector, and a method of preparing the peptide by using thetransformant.

The term “expression vector”, as used herein, refers to a recombinantvector capable of expressing a target peptide in a host cell, and refersto a genetic construct including essential regulatory elements which areoperably linked to express a gene insert. The expression vector includesexpression regulatory sequences such as an initiation codon, a stopcodon, a promoter, an operator, etc. The initiation and stop codons aregenerally considered as part of a nucleotide sequence encoding apolypeptide and are necessary to be functional in an individual to whoma genetic construct has been administered, and must be in frame with thecoding sequence. The promoter of the vector may be constitutive orinducible.

The term “operably linked”, as used herein, refers to a functionallinkage between a nucleic acid expression control sequence and anucleotide sequence encoding a target protein or RNA in such a manner asto allow general functions. For example, a promoter may be operablylinked to a nucleotide sequence encoding a protein or RNA to influencethe expression of the coding sequence. The operable linkage to theexpression vector may be prepared by using a recombinant genetictechnique well known in the art, and site-specific DNA cleavage andligation may be carried out by using enzymes generally known in the art.

Further, the expression vector may include signal sequences for thedischarge of the peptide in order to promote isolation of the peptidefrom a cell culture. Specific initiation signals may also be requiredfor efficient translation of inserted nucleotide sequences. Thesesignals include ATG initiation codon and adjacent sequences. In somecases, exogenous translational control signals, including ATG initiationcodon, should be provided. These exogenous translational control signalsand initiation codons may be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by theintroduction of appropriate transcription or translation enhancerelements.

In addition, the expression vector may further include a protein tagthat may be optionally removed by endopeptidase in order to facilitatethe detection of the peptide.

The term “tag”, as used herein, refers to a molecule which exhibits aquantifiable activity or characteristic. The tag may include fluorescentmolecules including chemical fluorescent such as fluorescein andpolypeptide fluorescent such as green fluorescent protein (GFP) orrelated proteins; and epitope tags such as a Myc tag, a Flag tag, aHis-tag, a leucine tag, an IgG tag, a streptavidin tag, etc. Inparticular, if an epitope tag is used, a peptide tag consisting ofpreferably 6 or more amino acid residues, and more preferably, about 8to 50 amino acid residues may be used.

In the present invention, the expression vector may include a nucleotidesequence encoding the above-described peptide for promoting regenerationof hard tissue, including dentin, bone, and cementum and/or dental pulpand treating dentin-dental pulp diseases and/or periodontal diseases ofthe present invention.

The vector used herein is not expressly limited, as long as it canproduce the peptide. Preferably, the vector may be plasmid DNA, phageDNA, etc. More preferably, the vector may be a commercially developedplasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid(pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derivedplasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24,YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, Agt10,Agtll, AZAP, etc.), an animal virus vector (retrovirus, adenovirus,vaccinia virus, etc.), an insect virus (baculovirus, etc.), or the like.

For the expression vector, a host cell most suitable for the intendeduse is preferably selected and used, because the expression level andmodification of protein vary depending on the kind of host cell.

The transformant of the present invention may be prepared bytransformation of a host with the expression vector of the presentinvention, and the transformant may express the polynucleotide in theexpression vector, thereby producing the peptide. Various methods mayperform the transformation. The transformation method is notparticularly limited, as long as it may produce the peptide. CaCl2precipitation, a Hanahan method that is an improved CaCl2 precipitationmethod by using DMSO (dimethyl sulfoxide) as a reducing agent,electroporation, calcium phosphate precipitation, protoplast fusion,agitation using silicon carbide fiber, Agrobacterium-mediatedtransformation, PEG-mediated transformation, dextran sulfate-,lipofectamine- or desiccation/inhibition-mediated transformation, etc.may be used. The host used in the preparation of the transformant is notparticularly limited, as long as it may produce the peptide of thepresent invention. The host may be bacterial cells such as E. coli,Streptomyces, Salmonella typhimurium, etc.; yeast cells such asSaccharomyces cerevisiae, Schizosaccharomyces pombe, etc.; fungal cellssuch as Pichia pastoris, etc.; insect cells such as Drosophila,Spodoptera Sf9 cells, etc.; animal cells such as CHO, COS, NSO, 293,Bowes melanoma cells, etc.; and plant cells.

The transformant may be used in a method of producing the peptide forpromoting regeneration of hard tissue, including dentin, bone, andcementum and/or dental pulp and treating dentin-dental pulp diseasesand/or periodontal diseases of the present invention. Specifically, themethod of producing the peptide for promoting regeneration of dentin ordental pulp and treating dentin or dental pulp diseases of the presentinvention may include (a) culturing the transformant to obtain aculture; and (b) recovering the peptide of the present invention fromthe culture.

The term “culturing”, as used herein, refers to a method of allowing amicroorganism to grow under artificially controlled environmentalconditions. In the present invention, the method of culturing thetransformant may be performed by a method widely known in the art.Specifically, the culturing is not particularly limited, as long as itmay express and produce the peptide for promoting regeneration of hardtissue including dentin, bone, and cementum and/or dental pulp andtreating dentin or dentin-dental pulp diseases and/or periodontaldiseases of the present invention, and the culturing may be performed bya batch process, a fed-batch process, or a repeated fed-batch process.

A medium used in the culturing includes appropriate carbon sources,nitrogen sources, amino acids, vitamins, etc. and should satisfy therequirements of a specific strain suitably while adjusting temperature,pH, etc. under aerobic conditions. Applicable carbon sources mayinclude, in addition to mixed sugars of glucose and xylose as a primarycarbon source, sugars and carbohydrates such as sucrose, lactose,fructose, maltose, starch, and cellulose, oils and fats such as soybeanoil, sunflower oil, castor oil, coconut oil, etc., fatty acids such aspalmitic acid, stearic acid, or linoleic acid, alcohols such as glycerolor ethanol, and organic acids such as acetic acid.

These substances may be used alone or in combination. Applicablenitrogen sources may include inorganic nitrogen sources such as ammonia,ammonium sulfate, ammonium chloride, ammonium acetate, ammoniumphosphate, ammonium carbonate, or ammonium nitrate; amino acids such asglutamic acid, methionine, or glutamine; and organic nitrogen sourcessuch as peptone, NZ-amine, meat extract, yeast extract, malt extract,steep corn liquor, casein hydrolysate, fish meal or digested productsthereof, defatted soybean cake or digested products thereof, etc. Thesenitrogen sources may be used alone or in combination. The medium mayinclude, as phosphorus sources, potassium phosphate monobasic, potassiumphosphate dibasic, and corresponding sodium-containing salts. Applicablephosphorus sources may include potassium dihydrogen phosphate,dipotassium hydrogen phosphate, or corresponding sodium-containingsalts. Also, inorganic compounds may include sodium chloride, calciumchloride, iron chloride, magnesium sulfate, iron sulfate, manganesesulfate, and calcium carbonate. In addition to the above materials,essential growth materials such as amino acids and vitamins may be used.

Further, appropriate precursors may be used in the culture medium.During culturing, the above-described materials may be appropriatelyadded to the culture in a batch, fed-batch, or continuous manner, butare not limited thereto. The pH of the culture may be adjusted byappropriately using a basic compound such as sodium hydroxide, potassiumhydroxide, or ammonia, or an acidic compound such as phosphoric acid orsulfuric acid.

In addition, the formation of bubbles may be inhibited by using anantifoaming agent such as fatty acid polyglycol ester.

In order to maintain an aerobic state, oxygen or oxygen-containing gas(e.g., air) may be injected into the culture. The temperature of theculture is generally 27° C. to 37° C., preferably 30° C. to 35° C.Culturing is continued until the desired level of the peptide productionwill be obtained. This is achieved within 10 hours to 100 hours.

In addition, the recovering of the peptide from the culture may beperformed by a method known in the art. Specifically, the recoveringmethod is not particularly limited, as long as it may recover theproduced peptide. Preferably, a method such as centrifugation,filtration, extraction, spraying, drying, evaporation, precipitation,crystallization, electrophoresis, fractional dissolution (e.g., ammoniumsulfate precipitation), chromatography (e.g., ion exchange, affinity,hydrophobic, and size exclusion), etc. may be used.

In still another aspect, the present invention provides a pharmaceuticalcomposition for preventing or treating dentin-dental pulp diseasesand/or periodontal diseases comprising the peptide.

As described above, when the peptide for promoting regeneration of hardtissue including dentin, bone and cementum and/or dental pulp andtreating dentin-dental pulp diseases and/or periodontal diseases of thepresent invention is transplanted into the body, together with humandental pulp cells, formation of dentin/dental pulp-like tissue by thehuman dental pulp cells may be promoted, and when the peptide is appliedto the damaged dentin or dental pulp site, the same physiologic dentinas observed in the natural human tooth dentin may be formed. Therefore,the peptide may be used as an active ingredient of the pharmaceuticalcomposition for treating dentin-dental pulp diseases, which are causedby damage to dentin or dental pulp.

The peptide included in the pharmaceutical composition may be used in asingle form of the peptide or in a polypeptide form of 2 or more repeatsof the peptide, and the peptide may also be used in a complex form of adrug having a therapeutic effect on dentin or dental pulp diseaseslinked at the N-terminus or C-terminus of the peptide.

The term “dentin-dental pulp diseases”, as used herein, refer to alldiseases caused by damaged dental pulp tissue and dentin linked to thedental pulp due to damage to the dentin and dental pulp tissues.

In the present invention, the dentin-dental pulp diseases are notparticularly limited, as long as the peptide of the present inventionexhibits the therapeutic effects on the diseases, and the dentin ordental pulp diseases may include, for example, dentin hypersensitivity,pulp hyperemia, pulpitis, pulp degeneration, pulp necrosis, gangrenouspulp, etc.

In still another aspect, the present invention provides a pharmaceuticalcomposition for preventing or treating periodontal diseases comprisingthe peptide. As described above, when the peptide for promotingregeneration of hard tissue including dentin, bone and cementum and/ordental pulp and treating dentin-dental pulp diseases and/or periodontaldiseases of the present invention is transplanted into the body,together with human dental pulp cells, formation of bone-like tissue bythe human dental pulp cells may be promoted. Therefore, the peptide maybe used as an active ingredient of the pharmaceutical composition fortreating periodontal diseases, which are caused by damage to bone and/orcementum.

The peptide included in the pharmaceutical composition may be used in asingle form of the peptide or in a polypeptide form of 2 or more repeatsof the peptide, and the peptide may also be used in a complex form of adrug having a therapeutic effect on dentin or dental pulp diseaseslinked at the N-terminus or C-terminus of the peptide.

The term “periodontal disease”, as used herein, also referred to aschronic periodontitis, refers to a disease that infects the periodontalligament and adjacent tissues by infection of bacteria in the gapbetween the gingiva and the teeth, depending on the severity of thedisease it is divided into gingivitis or periodontitis. During the onsetof periodontal disease, inflammation progresses, and more tissues aredamaged to form a periodontal pocket. It is known when periodontitisgets worse, and the periodontal pocket becomes deeper the periodontalpocket causes inflammation of periodontal ligament and finally causebone loss.

In the present invention, the periodontal diseases are not particularlylimited, as long as the peptide of the present invention exhibits thetherapeutic effects on the diseases, and the dentin or dental pulpdiseases may include, for example, gingivitis, periodontitis,periodontal pocket or periodontal abscess, etc.

The term “preventing”, as used herein, means all actions by which theoccurrence of dentin-dental pulp diseases is restrained or retarded byadministration of the pharmaceutical composition for preventing ortreating dentin-dental pulp diseases including the peptide of thepresent invention.

The term “treating”, as used herein, means all actions by which dentindental pulp diseases are treated by promoting regeneration of dentin ordental pulp by administering the pharmaceutical composition comprisingthe peptide of the present invention as an active ingredient to asubject in need of treatment of dentin-dental pulp diseases or allactions which are carried out by administering a pharmaceuticalcomposition comprising the peptide of the present invention as an activeingredient to an individual in need of treatment of periodontal diseaseby promoting regeneration of bone and/or cementum.

The pharmaceutical composition of the present invention may be preparedin the form of a pharmaceutical composition for treating dentin-dentalpulp diseases and/or periodontal diseases further including, in additionto the peptide, an appropriate carrier (natural or non-natural carrier),excipient, or diluent commonly used in the preparation of pharmaceuticalcompositions. Notably, the pharmaceutical composition may be formulatedaccording to a standard method in the form of a sterile injectablesolution that may be administered to dentin or dentin-dental pulpdiseases and/or periodontal diseases-induced site. In the presentinvention, the carrier, excipient, and diluent which may be included inthe pharmaceutical composition may include lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acaciarubber, alginate, gelatin, calcium phosphate, calcium silicate,cellulose, methylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oils, collagen, etc.Upon formulation, commonly used diluents or excipients such as a filler,an extender, a binder, a wetting agent, a disintegrant, a surfactant,etc. may be used. In particular, a sterilized aqueous solution, anon-aqueous solvent, a suspension, an emulsion, a freeze-driedpreparation, a suppository, an ointment (e.g., pulp liner, etc.) may beincluded. As non-aqueous solvents or suspensions, propylene glycol,polyethylene glycol, plant oils such as olive oil, injectable esterssuch as ethyl oleate, etc. may be used. As a base of the suppositories,witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin,etc. may be used.

A content of the peptide in the pharmaceutical composition of thepresent invention is not particularly limited, but the peptide may beincluded in an amount of 0.0001% by weight to 50% by weight, morepreferably, 0.01% by weight to 20% by weight, based on the total eightof the final composition.

The pharmaceutical composition of the present invention may beadministered in a pharmaceutically effective amount. The term“pharmaceutically effective amount”, as used herein, refers to an amountsufficient to treat or prevent diseases, at a reasonable benefit/riskratio applicable to any medical treatment or prevention. An effectivedosage level may be determined depending on factors including theseverity of the disease, drug activity, a patient's age, body weight,health conditions, sex, sensitivity to the drug, administration time,administration route, and excretion rate of the composition of thepresent invention, duration of treatment, drugs blended with orco-administered with the composition of the present invention, and otherfactors known in the medical field. The pharmaceutical composition ofthe present invention may be administered individually or in combinationwith a known pharmaceutical composition for treating dentin-dental pulpdiseases and/or periodontal diseases. It is essential to administer thecomposition in a minimum amount that may exhibit a maximum effectwithout causing side effects, because of all the above-describedfactors.

An administration dose of the pharmaceutical composition of the presentinvention may be determined by those skilled in the art, because of thepurpose of use, severity of the disease, a patient's age, body weight,sex, and medical history, a kind of a material used as an activeingredient, etc. For example, the pharmaceutical composition of thepresent invention may be administered at a dose of about 0.1 ng/kg toabout 100 mg/kg. Preferably, about 1 ng/kg to about 10 mg/kg per adultand administration frequency of the composition of the present inventionis not particularly limited. However, the composition may beadministered once a day or several times a day in divided doses. Theadministration dose does not limit the scope of the present invention inany aspect.

In still another aspect, the present invention provides a method oftreating dentin-dental pulp diseases, the method including administeringthe pharmaceutically effective amount of the pharmaceutical compositionto a human or a subject having dentin-dental pulp diseases, excludinghumans.

The term “subject, as used herein, may include mammals including humans,rats, livestock, etc. in need of treatment of dentin-dental pulpdiseases and/or periodontal diseases without limitation, but humans canbe excluded from the subjects having the above diseases.

The pharmaceutical composition for treating dentin-dental pulp diseasesand/or periodontal diseases of the present invention may be administeredvia any general route, as long as the pharmaceutical composition is ableto reach a target tissue.

The pharmaceutical composition may be administered, but is notparticularly limited to, via intraoral administration, intraoralinjection, etc., depending on the purpose.

In still another aspect, the present invention provides a quasi-drugcomposition for preventing or alleviating dentin-dental pulp diseases,including the peptide.

The term “alleviating”, as used herein, means all actions that at leastreduce a parameter related to the conditions to be treated, for example,the degree of symptom.

In the present invention, the alleviating is to be interpreted as allactions by which symptoms of dentin-dental pulp diseases have taken aturn for the better or been modified favorably by promoting regenerationof dentin-dental pulp or symptoms of periodontal diseases have taken aturn for the better or been modified favorably by promoting regenerationof bone and/or cementum by administering the pharmaceutical compositionincluding the peptide of the present invention as an active ingredientto a subject in need of treatment of dentin or dental pulp diseases.

The term “quasi-drug”, as used herein, refers to an article having amilder action than drugs, among articles being used for diagnosis,treatment, improvement, alleviation, handling, or prevention of human oranimal diseases. For example, according to Pharmaceutical Affairs Law,the quasi-drugs are those, excluding articles used as drugs, includingarticles made from fiber or rubber which are used to treat or preventhuman or animal diseases, articles, other than a tool or a machine, oran analog thereof, which have a mild action on or have no directinfluence on the human body, and articles which are used fordisinfection or pest control for the prevention of infectious diseases.

In the present invention, a kind of formulation of the quasi-drugcomposition including the peptide is not particularly limited, but thequasi-drug composition may be, for example, oral antiseptic mouthwashes,oral hygiene products, toothpastes, floss, oral ointments, etc.

In still another aspect, the present invention provides a healthfunctional food composition for preventing or alleviating dentin-dentalpulp diseases and/or periodontal diseases, including the peptide.

The term “food”, as used herein, includes meats, sausages, breads,chocolates, candies, snacks, confectionery, pizzas, ramen noodles, othernoodles, gums, dairy products including ice-creams, various soups,beverages, teas, drinks, alcoholic beverages, and vitamin complexes,health functional foods, health foods, etc., and the food includes allfoods in the ordinary acceptation of the term.

The term “functional food”, as used herein, is the term identical to thefood for special health use (FoSHU), and refers to a food having highmedical, medicinal effects, which is processed to exhibit thebiologically modulating function efficiently as well as to supplynutrients. Here, the term “functional” indicates a beneficial effect forhuman health, such as the regulation of nutrients for the structure andfunction of the human body, physiological action, etc. The food of thepresent invention may be prepared according to a method commonlyemployed in the art, and raw materials and ingredients commonly used inthe art may be added upon preparing the food. In addition, a formulationof the food is not limited, as long as the formulation is accepted as afood. The food composition of the present invention may be prepared as avariety of formulations. Since the food is used as raw materials, unlikegeneral drugs, the food composition lacks side effects which may occurwhen a drug is taken for a long period, and may have excellentportability. Therefore, the food of the present invention may be takenas a supplement for enhancing the effects of preventing or alleviatingdentin-dental pulp diseases and/or periodontal diseases.

The health food means food is having effects of actively maintaining orpromoting health conditions, as compared with general foods, and thehealth supplement food means a food for supplementing health. Ifnecessary, the health functional food, health food, and healthsupplement food may be interchangeably used.

Specifically, the health functional food is a food prepared by addingthe peptide of the present invention to food materials such asbeverages, teas, spices, gums, confectionery, etc., or prepared as acapsule, a powder, a suspension, etc. The functional health food meansthat it takes a specific effect on health when consumed, but unlikegeneral drugs, the health functional food has an advantage of having noside effects that may occur when a drug is taken for a long time,because it uses a food as a raw material.

Since the food composition of the present invention is routinelyingested, the food composition is expected to show high efficacy onprevention or improvement of dentin-dental pulp diseases and/orperiodontal diseases. Thus, it may be very usefully applied.

The food composition may further include a physiologically acceptablecarrier. A kind of the carrier is not particularly limited. Any carriermay be used, as long as it is commonly used in the art.

Further, the food composition may include additional ingredients thatare commonly used in food compositions to improve smell, taste, vision,etc. For example, the food composition may include vitamins A, C, D, E,B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc.Additionally, the food composition may also include minerals such as Zn,Fe, Ca, Cr, Mg, Mn, Cu, etc. Additionally, the food composition may alsoinclude amino acids such as lysine, tryptophan, cysteine, valine, etc.Additionally, the food composition may also include food additives, suchas preservatives (potassium sorbate, sodium benzoate, salicylic acid,sodium dehydroacetate, etc.), disinfectants (bleaching powder, higherbleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), coloring agents(tar color, etc.), color-developing agents (sodium nitrite, etc.),bleaching agents (sodium sulfite), seasonings (monosodium glutamate(MSG), etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.),flavors (vanillin, lactones, etc.), swelling agents (alum, potassiumD-bitartrate, etc.), fortifiers, emulsifiers, thickeners (adhesivepastes), film-forming agents, gum base agents, antifoaming agents,solvents, improvers, etc. The additives may be selected and used in anappropriate amount according to the food types.

The peptide of the present invention may be added as it is, or may beused in conjunction with other foods or food ingredients according to astandard method, or may be used appropriately according to a standardmethod. Mixing amounts of the active ingredient may be suitablydetermined depending upon the purpose of use (prophylactic, health ortherapeutic treatment). Generally, upon production of a food or abeverage, the food composition of the present invention may be added inan amount of 50 parts by weight or less, specifically 20 parts by weightor less, based on the total weight of the food or the beverage. However,when prolonged intake is intended for health and hygiene, the foodcomposition may be included in an amount below the above range. Inaddition, since there is no safety problem, the active ingredient may beused in an amount above the high range.

The food composition of the present invention may be used as, forexample, a health beverage composition. In this case, the healthbeverage composition may further include various flavors or naturalcarbohydrates, as in common beverages. The natural carbohydrates mayinclude monosaccharides such as glucose and fructose; disaccharides suchas maltose and sucrose; polysaccharides such as dextrin andcyclodextrin; and sugar alcohols such as xylitol, sorbitol, anderythritol. The sweeteners may be natural sweeteners such as thaumatinor a stevia extract; or synthetic sweeteners such as saccharine oraspartame. The natural carbohydrate may be generally used in an amountof about 0.01 g to 0.04 g, and specifically, about 0.02 g to 0.03 g,based on 100 mL of the health beverage composition of the presentinvention.

In addition, the health beverage composition may include variousnutrients, vitamins, minerals, flavors, colorants, pectic acid and saltsthereof, alginic acid and salts thereof, organic acids, protectivecolloidal thickeners, pH modifiers, stabilizers, antiseptics, glycerin,alcohols, carbonating agents, etc. Moreover, the health beveragecomposition may include the fruit flesh used to prepare natural fruitjuices, fruit juice beverages, or vegetable beverages. These ingredientsmay be used individually or in combination. A proportion of theadditives is not critical, but is generally selected from 0.01 parts byweight to 0.1 parts by weight per 100 parts by weight of the healthbeverage composition of the present invention.

The food composition of the present invention may include the peptide ofthe present invention in a variety of % by weight, as long as it mayexhibit the effect of preventing or alleviating dentin-dental pulpdiseases and/or periodontal diseases. Specifically, the peptide of thepresent invention may be included in an amount of 0.00001% by weight to100% by weight or 0.01% by weight to 80% by weight, based on the totalweight of the food composition, but is not limited thereto.

In still another aspect, the present invention provides a method ofpreventing or treating dentin-dental pulp diseases and/or periodontaldiseases, the method including administering the composition, includingthe peptide to a subject.

In still another aspect, the present invention provides a method ofpromoting regeneration of dentin or dental pulp tissues and/or hardtissue including dentin, bone, and cementum, the method includingadministering the composition including the peptide to a subject.

In still another aspect, the present invention provides use of a peptideincluding an amino acid sequence of the following Formula 1 or acomposition comprising the peptide in promoting regeneration of hardtissue including dentin, bone and cementum and/or dental pulp andtreating dentin-dental pulp diseases or periodontal diseases:

K-Y-R1-R2-R3-R4-R5   (Formula 1)

wherein R1 and R2 are arginine(R), lysine(K), glutamine(Q) orasparagine(N), respectively;

R3, R4, and R5 are arginine(R) or lysine(K), respectively.

In still another aspect, the present invention provides use of a peptideincluding any one amino acid sequence of SEQ ID

NOS: 1 to 128 or a composition including the peptide in promotingregeneration of hard tissue including dentin, bone and cementum and/ordental pulp and in treating dentin-dental pulp diseases and/orperiodontal diseases.

Hereinafter, the present invention will be described in more detail withreference to Examples. However, these examples are for illustrativepurposes only, and the scope of the present invention is not intended tobe limited by these Examples.

EXAMPLE 1 Methods and Materials

Synthesis of peptides for promoting generation of hard tissue includingdentin, bone and cementum and/or dental pulp and in treatingdentin-dental pulp diseases and/or periodontal diseases

The present inventors synthesized a peptide (SEQ ID NO: 1) showing theeffect of promoting regeneration of hard tissue including dentin, boneand cementum and/or dental pulp tissue by a 9-fluorenylmethyloxycarbonyl(Fmoc) method, and they synthesized peptides of Representative groups(Tables 1 to 16) by substituting the amino acids of the synthesizedpeptide.

(SEQ ID NO: 1) N-KYQRRKK-C

First, peptides of Group 1 were synthesized by using the peptide of SEQID NO: 1 or by substituting any amino acid at positions 5 to 7 of thepeptide of SEQ ID NO: 1 with lysine or arginine (Table 1).

TABLE 1  Peptides of Group 1 SEQ ID NO: Amino acid sequence (N-C) 1KYQRRKK 2 KYQRRKR 3 KYQRRRK 4 KYQRRRR 5 KYQRKKK 6 KYQRKRK 7 KYQRKKR 8KYQRKRR

Next, peptides of Group 2 were synthesized by substituting an amino acidat position 3 of the peptide of SEQ ID NO: 1 with arginine, bysubstituting an amino acid at position 4 of the peptide of SEQ ID NO: 1with glutamine, or by substituting any amino acid at positions 5 to 7 ofthe peptide of SEQ ID NO: 1 with lysine or arginine (Table 2).

TABLE 2  Peptides of Group 2 SEQ ID NO: Amino acid sequence (N-C)  9KYRQRKK 10 KYRQRKR 11 KYRQRRK 12 KYRQRRR 13 KYRQKKK 14 KYRQKRK 15KYRQKKR 16 KYRQKRR

Next, peptides of Group 3 were synthesized by substituting an amino acidat position 3 of the peptide of SEQ ID NO: 1 with lysine, bysubstituting an amino acid at position 4 of the peptide of SEQ ID NO: 1with glutamine, or by substituting any amino acid at positions 5 to 7 ofthe peptide of SEQ ID NO: 1 with lysine or arginine (Table 3).

TABLE 3  Peptides of Group 3 SEQ ID NO: Amino acid sequence (N-C) 17KYKQRKK 18 KYKQRKR 19 KYKQRRK 20 KYKQRRR 21 KYKQKKK 22 KYKQKRK 23KYKQKKR 24 KYKQKRR

Next, peptides of Group 4 were synthesized by substituting an amino acidat position 4 of the peptide of SEQ ID NO: 1 with glutamine, or bysubstituting any amino acid at positions 5 to 7 of the peptide of SEQ IDNO: 1 with lysine or arginine (Table 4).

TABLE 4  Peptides of Group 4 SEQ ID NO: Amino acid sequence (N-C) 25KYQQRKK 26 KYQQRKR 27 KYQQRRK 28 KYQQRRR 29 KYQQKKK 30 KYQQKRK 31KYQQKKR 32 KYQQKRR

Next, peptides of Group 5 were synthesized by substituting an amino acidat position 3 of the peptide of SEQ ID NO: 1 with arginine, or bysubstituting any amino acid at positions 5 to 7 of the peptide of SEQ IDNO: 1 with lysine or arginine (Table 5).

TABLE 5  Peptides of Group 5 SEQ ID NO: Amino acid sequence (N-C) 33KYRRRKK 34 KYRRRKR 35 KYRRRRK 36 KYRRRRR 37 KYRRKKK 38 KYRRKRK 39KYRRKKR 40 KYRRKRR

Next, peptides of Group 6 were synthesized by substituting an amino acidat position 3 of the peptide of SEQ ID NO: 1 with lysine, bysubstituting any amino acid at positions 5 to 7 of the peptide of SEQ IDNO: 1 with lysine or arginine (Table 6).

TABLE 6  Peptides of Group 6 SEQ ID NO: Amino acid sequence (N-C) 41KYKRRKK 42 KYKRRKR 43 KYKRRRK 44 KYKRRRR 45 KYKRKKK 46 KYKRKRK 47KYKRKKR 48 KYKRKRR

Next, peptides of Group 7 were synthesized by substituting an amino acidat position 4 of the peptide of SEQ ID NO: 1 with lysine, or bysubstituting any amino acid at positions 5 to 7 of the peptide of SEQ IDNO: 1 with lysine or arginine (Table 7).

TABLE 7  Peptides of Group 7 SEQ ID NO: Amino acid sequence (N-C) 49KYQKRKK 50 KYQKRKR 51 KYQKRRK 52 KYQKRRR 53 KYQKKKK 54 KYQKKRK 55KYQKKKR 56 KYQKKRR

Next, peptides of Group 8 were synthesized by substituting an amino acidat position 3 of the peptide of SEQ ID NO: 1 with asparagine, bysubstituting an amino acid at position 4 of the peptide of SEQ ID NO: 1with lysine, or by substituting any amino acid at positions 5 to 7 ofthe peptide of SEQ ID NO: 1 with lysine or arginine (Table 8).

TABLE 8  Peptides of Group 8 SEQ ID NO: Amino acid sequence (N-C) 57KYNKRKK 58 KYNKRKR 59 KYNKRRK 60 KYNKRRR 61 KYNKKKK 62 KYNKKRK 63KYNKKKR 64 KYNKKRR

Next, peptides of Group 9 were synthesized by substituting an amino acidat position 3 of the peptide of SEQ ID NO: 1 with asparagine, bysubstituting any amino acid at positions 5 to 7 of the peptide of SEQ IDNO: 1 with lysine or arginine (Table 9).

TABLE 9  Peptides of Group 9 SEQ ID NO: Amino acid sequence (N-C) 65KYNRRKK 66 KYNRRKR 67 KYNRRRK 68 KYNRRRR 69 KYNRKKK 70 KYNRKRK 71KYNRKKR 72 KYNRKRR

Next, peptides of Group 10 were synthesized by substituting an aminoacid at position 3 of the peptide of SEQ ID NO: 1 with arginine, bysubstituting an amino acid at position 4 of the peptide of SEQ ID NO: 1with asparagine, or by substituting any amino acid at positions 5 to 7of the peptide of SEQ ID NO: 1 with lysine or arginine (Table 10).

TABLE 10  Peptides of Group 10 SEQ ID NO: Amino acid sequence (N-C) 73KYRNRKK 74 KYRNRKR 75 KYRNRRK 76 KYRNRRR 77 KYRNKKK 78 KYRNKRK 79KYRNKKR 80 KYRNKRR

Next, peptides of Group 11 were synthesized by substituting an aminoacid at position 3 of the peptide of SEQ ID NO: 1 with lysine, bysubstituting an amino acid at position 4 of the peptide of SEQ ID NO: 1with asparagine, or by substituting any amino acid at positions 5 to 7of the peptide of SEQ ID NO: 1 with lysine or arginine (Table 11).

TABLE 11  Peptides of Group 11 SEQ ID NO: Amino acid sequence (N-C) 81KYKNRKK 82 KYKNRKR 83 KYKNRRK 84 KYKNRRR 85 KYKNKKK 86 KYKNKRK 87KYKNKKR 88 KYKNKRR

Next, peptides of Group 12 were synthesized by substituting an aminoacid at position 4 of the peptide of SEQ ID NO: 1 with asparagine, or bysubstituting any amino acid at positions 5 to 7 of the peptide of SEQ IDNO: 1 with lysine or arginine (Table 12).

TABLE 12  Peptides of Group 12 SEQ ID NO: Amino acid sequence (N-C) 89KYQNRKK 90 KYQNRKR 91 KYQNRRK 92 KYQNRRR 93 KYQNKKK 94 KYQNKRK 95KYQNKKR 96 KYQNKRR

Next, peptides of Group 13 were synthesized by substituting an aminoacid at position 3 of the peptide of SEQ ID NO: 1 with asparagine, bysubstituting an amino acid at position 4 of the peptide of SEQ ID NO: 1with glutamine, or by substituting any amino acid at positions 5 to 7 ofthe peptide of SEQ ID NO: 1 with lysine or arginine (Table 13).

TABLE 13  Peptides of Group 13 SEQ ID NO: Amino acid sequence (N-C)  97KYNQRKK  98 KYNQRKR  99 KYNQRRK 100 KYNQRRR 101 KYNQKKK 102 KYNQKRK 103KYNQKKR 104 KYNQKRR

Next, peptides of Group 2 were synthesized by substituting an amino acidat positions 3 and 4 of the peptide of SEQ ID NO:

1 with asparagine, by substituting any amino acid at positions 5 to 7 ofthe peptide of SEQ ID NO: 1 with lysine or arginine (Table 14).

TABLE 14  Peptides of Group 14 SEQ ID NO: Amino acid sequence (N-C) 105KYNNRKK 106 KYNNRKR 107 KYNNRRK 108 KYNNRRR 109 KYNNKKK 110 KYNNKRK 111KYNNKKR 112 KYNNKRR

Next, peptides of Group 15 were synthesized by substituting an aminoacid at position 3 of the peptide of SEQ ID NO: 1 with arginine, bysubstituting an amino acid at position 4 of the peptide of SEQ ID NO: 1with lysine, or by substituting any amino acid at positions 5 to 7 ofthe peptide of SEQ ID NO: 1 with lysine or arginine (Table 15).

TABLE 15  Peptides of Group 15 SEQ ID NO: Amino acid sequence (N-C) 113KYRKRKK 114 KYRKRKR 115 KYRKRRK 116 KYRKRRR 117 KYRKKKK 118 KYRKKRK 119KYRKKKR 120 KYRKKRR

Lastly, peptides of Group 16 were synthesized by substituting an aminoacid at positions 3 and 4 of the peptide of SEQ ID NO: 1 with lysine, bysubstituting any amino acid at positions 5 to 7 of the peptide of SEQ IDNO: 1 with lysine or arginine (Table 16).

TABLE 16  Peptides of Group 16 SEQ ID NO: Amino acid sequence (N-C) 121KYKKRKK 122 KYKKRKR 123 KYKKRRK 124 KYKKRRR 125 KYKKKKK 126 KYKKKRK 127KYKKKKR 128 KYKKKRR

EXAMPLE 1-2 Cell Culture

Cells were cultured in humidified air containing about 37% of CO₂ at 37° C. Moreover, they were used in the experiment. Human bone marrowmesenchymal stem cells (hBMSCs) were purchased and used from Lonza(LONZA, Switzerland). hBMSCs were cultured in an alpha-MEM(a-MEM)(Invitrogen) culture medium containing 10% heat-inactivated bovineserum.

EXAMPLE 1-3 Separation and Culture of Human-Derived Dental Pulp Cells

Human dental pulp cells were separated from wisdom teeth of adults (aged18-22) at the School of Dentistry, Seoul National University. In detail,all experiments were performed after the approval of the InstitutionalReview Board and the informed consent from patients. Wisdom teeth werefractured according to a method of Jung HS et al. (J Mol Histol.(2011))to expose the dental pulps, and dental pulp tissues were separated withforceps. Each of the separated dental pulp tissues was cut into smallpieces with a razor blade, put in a 60-mm dish, covered with acoverslip, and then cultured in a Dulbecco's modified Eagle's medium. Ithas been known that human dental pulp cells can differentiate intoodontoblast, osteoblast, cementoblast, and periodontal ligament cellsunder various conditions (Tissue Eng Part A. 2014 Apr; 20 (7-8):1342-51).

EXAMPLE 1-4 Analysis of Reverse Transcription-Polymerase Chain Reaction(RT-PCR) and Real-Time PCR

Total RNA was extracted from human dental pulp cells (hDPCs), and humanbone marrow mesenchymal stem cells (hBMSCs) with TRIzol reagent. 2 μg ofthe total RNA, 1 pl of reverse transcriptase, and 0.5 μg of oligo(oligo; dT) were used to synthesize cDNA. The synthesized cDNA was usedin a real-time polymerase chain reaction. The real-time polymerase chainreaction was performed on an ABI PRISM 7500 sequence detection system(Applied Biosystems) and an SYBR GREEN PCR Master Mix (Takara, Japan).The real-time polymerase chain reaction was performed under conditionsof 94° C., 1 min; 95° C., 15 sec; 60° C., sec for 40 cycles. Resultswere analyzed by a comparative cycle threshold (CT) method. And the usedprimers are as follows (Table 17).

TABLE 17  <Complete lists of human real-time PCR primers> GenePrimer (5′-3′) hDspp Forward CAACCATAGAGAAAGCAAACGCG ReverseTTTCTGTTGCCACTGCTGGGAC hNestin forward AGCCCTGACCACTCCAGTTTAG reverseCCCTCTATGGCTGTTTCTTTCTCT hBSP forward GAATGGCCTGTGCTTTCTCAA reverseTCGGATGAGTCACTACTGCCC hGAPDH forward CCATGGAGAAGGCTGGGG reverseCAAAGTTCTCATGGATGACC

EXAMPLE 1-5 In Vivo Transplantation and Histomorphological Analysis

Human dental pulp cells (hDPCs) were isolated and used for in vivotransplantation experiments. Human dental pulp cells (2×10⁶) were mixedwith 100 mg of hydroxy apatite/tricalcium phosphate (HA/TCP) ceramicpowder (Zimmer, USA) alone, or with the peptide of the invention (10 μg)with 0.5% fibrin gel respectively, and then the prepared implanttransplanted to a mice with compromised immune systems (NIH-bg-nu-xid;Harlan Laboratories, Indianapolis, Ind.), and the mice were raised for 6and 12 weeks.

After that, the sample tissues were harvested and fixed in 4%paraformaldehyde, decalcified in 10% EDTA (pH 7.4), embedded inparaffin, stained with hematoxylin-eosin (H-E) (Vector Labs), orconducted Immunohistochemical analysis. To immunohistochemical analysis,proteins were detected with anti-DSP antibody diluted 1:150 as theprimary antigen, and goat anti-rabbit IgG (Vector Labs) labeled withbiotin as secondary antigen.

Collagen staining was conducted by using a Masson's Trichrome Stain Kit(Cat. 25088-100) of Polysciences, co.

Quantitative analysis of newly formed hard tissue was analyzed using theLS starter program (OLYMPUS Soft Imaging Solution, Muster, Germany). Theproportion of newly formed hard tissue was calculated as the percentageof the area of newly formed hard tissue in the total area.

EXAMPLE 1-6 Scanning Electron Microscope Analysis

The sample tissues were fixed in 2.5% Glutaraldehyde/0.1 M Cacodylatebuffer for 30 minutes and reacted in a solution containing 1% osmiumtetroxide in 0.1 M Cacodylate buffer for 1 hour. Then, the sampletissues were quickly dehydrated and dried using ethanol, and then thesample tissues were coated with gold and observed with a scanningelectron microscope (S-4700, HITACHI, Tokyo, Japan).

EXAMPLE 1-7 Statistical Analysis

Statistical analysis was performed using Student's t-test. Allstatistical analysis is performed by SPSS software ver. 19.0.

EXAMPLE 2 Experimental Results EXAMPLE 2-1

The effect of the peptides for the promotion of dentin or dental pulptissue and the treating of dentin or dental pulp diseases on theexpression level of odontoblast differentiation marker Dspp gene

The Dspp gene is used as a marker for odontoblast cell differentiationand is known as an essential gene for dentin calcification. Therefore,it was confirmed that the peptide of the present invention has an effectof promoting the expression of the Dspp gene, which is odontoblastdifferentiation marker gene, and promoting odontoblast and the formationof dentin.

The human dental pulp cells (hDPCs) cultured in Example 3 were treatedwith the peptides (concentration of 10 μg/ml) of each group synthesizedin Example 1-1, and cultured for 48 hours. Then, mRNA levels of anodontoblast differentiation marker Dspp gene expressed in the humandental pulp cells were measured, and a ratio of the measured Dspp mRNAlevel relative to a Dspp mRNA level measured in a control group wascalculated, respectively (Tables 18 to 33).

And, the average value of mRNA levels of the Dspp gene measuredaccording to the peptides of each group of Tables 1 to 3 was comparedfor each group (FIG. 1A). Specifically, the new peptides of the presentinvention lacking the substitution or partial sequence of amino acidnucleotide sequences are grouped as shown in Tables 1 to 3, and the newpeptides of each group are used for expression of Dspp, a blast celldifferentiation marker gene, in human dental pulp cells. As a result,showing the effect, a graph showing the average value of each groupmeasured by quantitative real-time PCR of the level of Dspp mRNA inhuman dental pulp cells is shown in FIG. 1A. In this case, human dentalpulp cells that were not treated with the peptide of the presentinvention were used as a control.

In addition, the average value of mRNA levels of the Dspp gene measuredaccording to the peptides of each group of Tables 4 to 16 was comparedfor each group (FIG. 1B). Specifically, the new peptides of the presentinvention lacking the substitution or partial sequence of amino acidbase sequences are grouped as shown in Tables 4 to 16, and the newpeptides of each group are expressed in the expression of Dspp, a blastcell differentiation marker gene, in human dental pulp cells. As aresult, showing the effect, a graph showing the average value of eachgroup measured by quantitative real-time PCR of the level of Dspp mRNAin human dental pulp cells is shown in FIG. 1B. In this case, humandental pulp cells that were not treated with the peptide of the presentinvention were used as a control.

The expression level of the Dspp gene was measured through RT-PCR andreal-time PCR analysis described in Example 1-4. In this regard, GAPDHgene was used as an internal control. The experiments were performed intriplicate, and then mean values and standard deviations thereof weretaken as measured values. The base sequence of the primers is describedin Table 17 above.

TABLE 18 Effects of peptides of group 1 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 1 7.371 0.093 27.171 0.121 3 6.512 0.209 4 7.071 0.192 5 6.893 0.07 6 6.931 0.119 76.881 0.321 8 6.531 0.2025

TABLE 19 Effects of peptides of group 2 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 9 7.543 0.132 106.996 0.352 11 7.385 0.271 12 7.548 0.327 13 6.655 0.377 14 6.839 0.24115 6.764 0.289 16 7.739 0.357

TABLE 20 Effects of peptides of group 3 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 17 7.712 0.219 187.319 0.192 19 7.931 0.192 20 7.553 0.299 21 7.893 0.132 22 7.412 0.37223 9.171 0.381 24 8.512 0.411

TABLE 21 Effects of peptides of group 4 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 25 2.491 0.453 262.623 0.273 27 2.213 0.302 28 2.781 0.5 29 2.926 0.292 30 2.011 0.311 312.432 0.52 32 2.303 0.299

TABLE 22 Effects of peptides of group 5 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 33 3.615 0.53 343.727 0.495 35 3.017 0.293 36 3.256 0.444 37 3.303 0.671 38 2.099 0.50639 3.412 0.279 40 3.109 0.395

TABLE 23 Effects of peptides of group 6 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 41 2.937 0.333 422.808 0.501 43 2.435 0.432 44 2.517 0.296 45 3.051 0.433 46 2.733 0.19847 2.439 0.287 48 2.602 0.333

TABLE 24 Effects of peptides of group 7 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 49 1.631 0.137 501.803 0.208 51 1.569 0.111 52 1.949 0.327 53 1.422 0.09 54 1.638 0.21455 2 0.396 56 1.909 0.55

TABLE 25 Effects of peptides of group 8 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 57 2.415 0.375 582.677 0.601 59 2.463 0.222 60 2.089 0.163 61 1.909 0.307 62 2.752 0.48263 2.373 0.394 64 1.829 0.201

TABLE 26 Effects of peptides of group 9 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 65 2.201 0.461 662.072 0.366 67 2.452 0.509 68 2.343 0.419 69 1.899 0.382 70 1.947 0.24771 2.052 0.233 72 1.739 0.188

TABLE 27 Effects of peptides of group 10 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 73 2.208 0.366 742.105 0.273 75 2.624 0.522 76 2.394 0.432 77 1.939 0.337 78 2.109 0.15979 2.403 0.601 80 2.636 0.573

TABLE 28 Effects of peptides of group 11 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 81 1.757 0.372 821.909 0.269 83 2.001 0.227 84 2.101 0.373 85 1.838 0.401 86 1.736 0.31787 1.888 0.444 88 1.539 0.132

TABLE 29 Effects of peptides of group 12 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 89 1.635 0.214 901.797 0.323 91 1.913 0.333 92 1.498 0.111 93 1.892 0.274 94 1.487 0.09995 1.939 0.295 96 2.011 0.199

TABLE 30 Effects of peptides of group 13 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 97 1.515 0.107 981.479 0.106 99 1.737 0.207 100 1.599 0.166 101 1.674 0.109 102 1.8550.299 103 1.737 0.107 104 1.878 0.201

TABLE 31 Effects of peptides of group 14 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 105 1.664 0.085106 1.673 0.207 107 1.935 0.372 108 1.495 0.091 109 1.756 0.201 1101.595 0.099 111 1.918 0.175 112 1.699 0.143

TABLE 32 Effects of peptides of group 15 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 113 1.778 0.205114 1.849 0.337 115 1.707 0.199 116 1.693 0.075 117 1.929 0.193 1182.015 0.151 119 2.121 0.337 120 1.878 0.116

TABLE 33 Effects of peptides of group 16 on mRNA level of Dspp gene mRNAlevel of Dspp gene SEQ ID NO: Mean Standard deviation 121 2.024 0.298122 1.979 0.303 123 1.837 0.111 124 2.017 0.402 125 2.082 0.377 1261.798 0.163 127 1.888 0.099 128 1.765 0.375

FIG. 1A is a graph showing the results of comparing the expressionlevels of the dentin sialophosphoprotein (Dspp), odontoblastdifferentiation marker gene, in human dental pulp cells (hDPCs) treatedwith the novel peptide of the present invention. Refer to FIG. 1A andTable 18 to Table 20, compared to the mRNA level of the Dspp gene, anodontoblast differentiation marker, measured in human dental pulp cells(control) not treated with the peptide of the present invention, whenthe peptide of the present invention is treated, it can be seen that allof the mRNA levels of the Dspp gene increased by about 6 to 8 times.Especially when treated with the peptide of group 3, it showed thehighest Dspp mRNA expression value. FIG. lB is another graph showing theresult of comparing the expression levels of Dspp, odontoblastdifferentiation marker gene, in human dental pulp cells (hDPCs) treatedwith the novel peptide of the present invention. Refer to FIG. 1B andTable 21 to Table 33, compared to the mRNA level of the Dspp gene, amarker of differentiation of odontoblast differentiation measured inhuman dental pulp cells (control) without treatment of the peptide ofthe present invention, when the peptide of the present invention istreated, it can be seen that all of the mRNA levels of the Dspp geneincreased by about 1.5 to 3 times.

EXAMPLE 2-2 Effects of Peptides for Promoting Regeneration of Dentin orDental Pulp Tissues and Treating Dental Pulp Diseases on ExpressionLevels of Odontoblast Differentiation Marker Gene, Nestin

The results of Example 2-1 showed that the peptides of the presentinvention might increase the Dspp mRNA level, for example, all groups ofpeptides can increase the mRNA level of the Dspp gene by more than 1.5times, and even more than 3 times, and in particular, the peptides ofGroup 1 and Group 3 may increase the Dspp mRNA level at least 6 times orhigher.

Accordingly, it was examined whether the peptides of Group and Group 3may increase mRNA levels of other odontoblast differentiation markergenes, Nestin.

Briefly, experiments were performed in the same and similar manner as inExample 2-1, except that the following primers were used. The effects ofthe peptides of the present invention on expression levels of Nestingenes were measured, and the calculated mean values were comparedbetween the groups (FIG. 1C). In this regard, human dental pulp cellsthat were treated with none of the peptides of the present inventionwere used as a control group.

FIG. 1C is a graph showing the results of comparing the expressionlevels of Nestin, odontoblast differentiation marker genes, in humandental pulp cells (hDPCs) treated with the peptide of the presentinvention. As shown in FIG. 1C, the group treated with the peptide ofthe present invention (Group 1, 2, 3) compared to the control group, itcan be seen that the expression level of the Nestin gene, which is amarker of differentiation of the odontoblast differentiation, isincreased by 5 times or more.

The above Dspp and Nestin genes are known as genes involved inodontoblast differentiation and dentin mineralization, which infers thatthe peptides of the present invention may exhibit the effect ofpromoting dentin regeneration.

EXAMPLE 2-3 Effect of Osteoblasts and/or Cementoblasts Promotion andPeriodontal Disease Treatment Peptide on the Expression Levels of BSPGene, Osteoblasts and Cementoblasts Differentiation Marker Gene

The BSP gene is used as a marker for differentiating osteoblasts andcementoblasts, and is known as an essential gene for calcification ofbone and cementum. Therefore, in order to confirm the effect of thenovel peptide of the present invention on the expression of the BSPgene, bone stem cell and a cementum cell differentiation marker gene,human-derived mesenchymal stem cells cultured by performing the methodof Example 1-2 above (after treating each group of peptides in humanbone marrow mesenchymal stem cells (hBMSCs)), BSP gene expression wasconfirmed by real-time PCR.

Briefly, except for using a different primer, the same and similarmethods as in Example 2-1 were performed to measure the effect of thepeptide of the present invention on the expression level of the BSPgene, and was measured for each group. The average level was compared(FIG. 2A, FIG. 2B). Specifically, in the novel peptides of the presentinvention grouped as shown in Tables 1 to 3, each group of new peptidesexpresses bone and cementum differentiation marker gene BSP (Bonesialoprotein) in human-derived mesenchymal stem cells (hBMSCs). As aresult, showing the effect, the result of measuring the level of BSPmRNA in human-derived mesenchymal stem cells by quantitative real-timePCR is shown in FIG. 2A. In addition, in the novel peptides of thepresent invention grouped as shown in Tables 4 to 16, as a resultshowing the effect of each group of new peptides on the expression ofBSP, a bone and cementum differentiation marker gene, in human-derivedmesenchymal stem cells, the result of measuring the level of BSP mRNA inthe derived mesenchymal stem cells by quantitative real-time PCR isshown in FIG. 2B.

At this time, the peptide was treated at a concentration of 10 μg/ml.And as a control, human bone marrow mesenchymal stem cells not treatedwith the peptide of the present invention were used.

FIG. 2A is a graph showing the results of comparing the expression levelof the BSP gene, a bone and cementum differentiation marker gene, inhuman-derived mesenchymal stem cells (hBMSCs) treated with the peptideof the present invention. As shown in FIG. 2A, the group treated withthe peptide of the present invention (Group 1, 2, 3), it can be seenthat the BSP gene expression increased by about 9 to 13 times or morecompared to the control. In particular, when treated with the peptide ofgroup 3, it showed the highest BSP mRNA expression value.

FIG. 2B is a graph showing the results of comparing the expression levelof the BSP gene, a bone and cementum differentiation marker gene, inhuman-derived mesenchymal stem cells (hBMSCs) treated with the peptideof the present invention. As shown in FIG. 2B, the group treated withthe peptide of the present invention (Group 4 to Group 16), can be foundthat the BSP gene expression is increased by about 3 to 9 times or more,and 12 times or more, compared to the control. In particular, whentreated with the peptide of group 11, it showed the highest BSP mRNAexpression value.

As the BSP gene is used as a marker for differentiating osteoblasts andcementoblasts and is known as a gene involved in the process ofcalcification of bone and cementum, it was analyzed that the peptideprovided in the present invention would have an effect of promotingregeneration of bone and cementum.

EXAMPLE 2-4 Hard Tssue Formation of Human Dental Pulp Cells (hDPCs) byNovel Peptides In Vivo for 6 Weeks (1) Histomorphological analysis

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 2A, and FIG. 2B, based on the results ofthe in vitro experiments, in order to measure the effect of the peptideof the present invention on hard tissue formation in vivo, described inExamples 1-5 above As described, human dental pulp cells (hDPCs) and 100mg of hydroxyapatite/tricalcium phosphate (HA/TCP) were mixed with 0.5μg fibrin gel, respectively, with 10 μg of group 3 peptides (eg, SEQ IDNO: 24) to prepare an implant. The implant was transplanted into thesubcutaneous tissue of a mouse with a compromised immune system. At thistime, as a control, a transplanted implant containing no peptide of thepresent invention was used. After 6 weeks of transplantation, asdescribed in Example 1-5 above, a sample was taken and then the newlyformed hard tissue was quantitatively analyzed using the LS starterprogram, and the results are shown in FIG. 3.

FIG. 3 shows the results of measuring the amount of newly formed hardtissue using human dental pulp cells (hDPCs) for 6 weeks in vivo. Asshown in FIG. 3, the ratio of hard tissue formation after 6 weeks oftransplantation was increased by about 2 times or more in the grouptreated with the novel peptide (Group 3, 29.6%) compared to the control(Control, 13.5%).

FIG. 4 is a microscopic image showing the histomorphological analysis ofthe hard tissue formed using human dental pulp cells (hDPCs) for 6 weeksin vivo, A to D show the results of transplantation of a control implantprepared by mixing hDPCs and 100 mg HA/TCP in a 0.5% fibrin gel for 6weeks in a mouse with compromised immune systems and E to H show theresults of transplantation of hDPCs and 100 mg HA/TCP with 0.5% fibringel, respectively, together with 10 μg of group 3 peptide, in a mousewith compromised immune systems for 6 weeks (scale bar: A, E 500 μm; B,F 200 μm; C, G 100 μm; D, H 50 μm).

As shown in FIG. 4, as a result of histomorphological analysis throughhematoxylin-eosin staining, in the control group (FIG. 4A to FIG. 4D)not containing the peptide of the present invention and a groupcontaining the peptide of the present invention (FIG. 4E to FIG. 4H)were observed that bone-like tissue and dentin-pulp-like tissue wereformed in the substrate of the calcified tissue around the HA/TCPparticles.

(2) Collagen Staining Analysis

Collagen is the most abundant organic matrix in dentin, bone andcementum, and serves to accommodate deposited minerals. Accordingly,collagen staining was performed to confirm the accumulation of collagenprotein in the calcified tissue formed in each experimental group of thehistomorphological analysis.

FIG. 5 shows microscopic images showing the level of collagen formationin hard tissue formed using human dental pulp cells (hDPCs) for 6 weeksin vivo, A to D show the results of transplantation of a control implantprepared by mixing hDPCs and 100 mg HA/TCP in a 0.5% fibrin gel for 6weeks in a mouse with compromised immune systems and E to H show theresults of transplantation of hDPCs and 100 mg HA/TCP with 0.5% fibringel, respectively, together with 10 μg of group 3 peptide, in a mousewith compromised immune systems for 6 weeks (scale bar: A, E 500 μm; B,F 200 μm; C, G 100 μm; D, H 50 μm). The formed hard tissue was stainedby the method of collagen stain (Masson's trichrome stain).

As shown in FIG. 5, compared to the control group (FIG. 5A to 5D), inthe group containing the peptide of the present invention (FIG. 5E to5H), it was confirmed that the formation level of collagen wasincreased.

(3) Immunohistochemical Analysis

The expression of DSP, odontoblast specific differentiation marker gene,was confirmed by immunohistochemical analysis.

FIG. 6 is an immunostaining picture showing the analysis of theexpression level of DSP, a marker for differentiation of blast cellsusing immunostaining method, in hard tissue formed using human dentalpulp cells (hDPCs) for 6 weeks in vivo, A to D show the results oftransplantation of a control implant prepared by mixing hDPCs and 100 mgHA/TCP in a 0.5% fibrin gel for 6 weeks in a mouse with compromisedimmune systems and E to H show the results of transplantation of hDPCsand 100 mg HA/TCP with 0.5% fibrin gel, respectively, together with 10μg of group 3 peptide, in a mouse with compromised immune systems for 6weeks. A and B were immunostained using the anti-DSP antibody. C is anegative control of immunohistochemical analysis treated with onlysecondary antibodies. Arrows marked A and B indicate the expression ofDSP in newly formed calcified tissue. The scale bar is 50 μm.

As shown in FIG. 6, the control group (FIG. 6A) was weakly expressed inDSP in newly formed dentin-pulp-like tissue, but the calcified tissue inwhich DSP was newly formed in the group containing the peptide of thepresent invention (FIG. 6B) was strongly expressed. FIG. 6C shows thatin the immunohistochemical analysis, the secondary antibody-treatednegative control group was not stained with DSP.

EXAMPLE 2-5 Hard Tissue Formation of Human Dental Pulp Cells (hDPCs) byNovel Peptides In Vivo for 12 Weeks

Except for raising the implanted mouse for 12 weeks, the method ofExample 2-4 was performed to analyze hard tissue formation in humandental pulp cells.

FIG. 7 shows the results of measuring the amount of newly formed hardtissue using human dental pulp cells (hDPCs) for 12 weeks in vivo. Asshown in FIG. 7, the ratio of hard tissue formation after 12 weeks oftransplantation was increased by about 2 times or more in the grouptreated with the novel peptide (Group 3, 39.5%) compared to the control(Control, 23.7%).

FIG. 8 is a microscopic image showing the histomorphological analysis ofthe hard tissue formed using human dental pulp cells (hDPCs) for 12weeks in vivo, A to D show the results of transplantation of a controlimplant prepared by mixing hDPCs and 100 mg HA/TCP in a 0.5% fibrin gelfor 12 weeks in a mouse with compromised immune systems and E to

H show the results of transplantation of hDPCs and 100 mg HA/TCP with0.5% fibrin gel, respectively, together with 10 μg of group 3 peptide,in a mouse with compromised immune systems for 12 weeks (scale bar: A, E500 μm; B, F 200 μm; C, G 100 μm; D, H 50 μm).

As shown in FIG. 8, as a result of histomorphological analysis throughhematoxylin-eosin staining, similar to the case of FIG. 4 (6 weekstransplant) in the control group (FIG. 8A to FIG. 8D) not containing thepeptide of the present invention and a group containing the peptide ofthe present invention (FIG. 8E to FIG. 8H) were observed that bone-liketissue and dentin-pulp-like tissue were formed in the substrate of thecalcified tissue around the HA/TCP particles.

(2) Collagen Staining Analysis

Collagen staining was performed to confirm the accumulation of collagenprotein in the calcified tissue formed in each experimental group of thehistomorphological analysis of Example 2-5.

FIG. 9 shows microscopic images showing the level of collagen formationin hard tissue formed using human dental pulp cells (hDPCs) for 12 weeksin vivo, A to D show the results of transplantation of a control implantprepared by mixing hDPCs and 100 mg HA/TCP in a 0.5% fibrin gel for 12weeks in a mouse with compromised immune systems and E to H show theresults of transplantation of hDPCs and 100 mg HA/TCP with 0.5% fibringel, respectively, together with 10 μg of group 3 peptide, in a mousewith compromised immune systems for 12 weeks (scale bar: A, E 500 μm; B,F 200 μm; C, G 100 μm; D, H 50 μm). The formed hard tissue was stainedby the method of collagen stain (Masson's trichrome stain).

As shown in FIG. 9, compared to the control group (FIG. 9A to 9D), inthe group containing the peptide of the present invention (FIG. 9E to9H), it was confirmed that the formation level of collagen wasincreased.

(3) Immunohistochemical Analysis

The expression of DSP, odontoblast specific differentiation marker gene,was confirmed by immunohistochemical analysis.

FIG. 10 is an immunostaining picture showing the analysis of theexpression level of DSP, a marker for differentiation of blast cellsusing immunostaining method, in hard tissue formed using human dentalpulp cells (hDPCs) for 12 weeks in vivo, A to D show the results oftransplantation of a control implant prepared by mixing hDPCs and 100 mgHA/TCP in a 0.5% fibrin gel for 12 weeks in a mouse with compromisedimmune systems and E to H show the results of transplantation of hDPCsand 100 mg HA/TCP with 0.5% fibrin gel, respectively, together with 10μg of group 3 peptide, in a mouse with compromised immune systems for 6weeks. A and B were immunostained using the anti-DSP antibody. C is anegative control of immunohistochemical analysis treated with onlysecondary antibodies. Arrows marked A and B indicate the expression ofDSP in newly formed calcified tissue. The scale bar is 50 μm.

As shown in FIG. 10, the control group (FIG. 10A) was weakly expressedin DSP in newly formed dentin-pulp-like tissue, but the calcified tissuein which DSP was newly formed in the group containing the peptide of thepresent invention (FIG. 10B) was strongly expressed. FIG. 10C shows thatin the immunohistochemical analysis, the secondary antibody-treatednegative control group was not stained with DSP.

Summarizing the results of Examples 2-4 and 2-5, it was found that thenovel peptide of the present invention exhibits an effect capable ofpromoting regeneration of dentin/pulp tissue complexes andbone/cementum-like tissues.

EXAMPLE 2-6 Cell Analysis using Scanning Electron Microscope ofTransplanted Tissue

Scanning electron microscope analysis of the method of Example 1-6 wasperformed to confirm the differentiation of human dental pulp cells(hDPCs) into odontoblast or osteoblast/cementoblast in the control groupand the experimental group treated with the novel peptide for 12 weeksafter transplantation was performed.

After 12 weeks, scanning electron microscope analysis was performed bythe method of Example 1-6 in order to confirm the differentiation ofhuman dental pulp cells (hDPCs) into odontoblast orosteoblast/cementoblast between the experimental group treated with thenovel peptide and the control group.

FIG. 11 is an image showing analysis of hard tissue formed using humandental pulp cells (hDPCs) for 12 weeks in vivo using a scanning electronmicroscope (SEM), wherein A is hDPCs and 100 mg HA/TCP was prepared bymixing 0.5% fibrin gel with control implant, B and C were hDPCs and 100mg HA/TCP with 10 μg of group 3 peptides, respectively, mixed with 0.5%fibrin gel. It shows the result of the implantation of the implant in amouse with a compromised immune system for 12 weeks. The scale bar is 10μm. The formed hard tissue was observed by cells using a scanningelectron microscope.

In the control group treated with hDPCs-alone, some of theodontoblast-like cells with incomplete odontoblastic processes wereformed around the formed hard tissue (FIG. 11A). In the group treatedwith the peptide of the present invention (for example, the peptide ofgroup 3), odontoblast-like cells were observed along the formed hardtissue and the odontoblastic processes were also extended toward theformed hard tissue (FIG. 11B). In addition, in the group treated withthe peptide of the present invention, it was confirmed that it exhibitsthe characteristics of typical osteoblasts/cementoblasts with cubicshape attached to the surface of the formed hard tissue (FIG. 11C).

Therefore, it was found that the peptide of the present invention canmore effectively form odontoblast and osteoblast/cementoblast.

EXAMPLE 2-7 Obturation Test of Dentinal Tubules In Vivo

In the premolar of a 12-month-old adult dog, a dental bur was used toremove the enamel of the cervical region and expose the dentin. Thepremolar of exposed dentin was sufficiently washed to completely removethe enamel-dentin fragments generated during vortex formation, followedby removal of moisture.

1.5 μg of the peptide (SEQ ID NO: 24) (group 3) according to the presentinvention was applied to the inlet of the dentinal tubule on the exposeddentin site, and after 3 weeks, the adult dog was euthanized to extractteeth. Then, a specimen of the extracted tooth was prepared using adiamond saw.

And in order to confirm the effect of the novel peptide according to thepresent invention on the exposed dentinal tubule obturation of thedamaged dentin, the ability to close the dentinal tubule was evaluatedthrough a scanning electron microscope and the results are shown in FIG.12.

Specifically, as shown in FIG. 12A and FIG. 12E, after cutting the lowerportion of the dentin injury site, the lower surface (box portion) ofthe cut surface was observed. As a result of the scanning electronmicroscope, it was confirmed that the control group without anytreatment exposed the dentinal tubules of the lower part of the damageddentin (FIG. 12A to FIG. 12D). On the other hand, in the experimentalgroup treated with peptides, it can be seen that the exposed dentinaltubules were closed by physiological remineralization (FIG. 12E to FIG.12H).

EXAMPLE 2-8 Observation of the Damaged Site of Dentin Surface In Vivo

A specimen of teeth extracted from adult dogs was prepared by the samemethod as in Example 2-7.

And to confirm the effect of the novel peptide according to the presentinvention on the dentinal tubule obturation on the surface of thedamaged dentin, the ability to close the dentin tubules on the surfacearea was evaluated through a scanning electron microscope, and theresults are shown in FIG. 13.

As a result of observation by scanning electron microscope, it can beconfirmed that the dentinal tubules are exposed on the damaged dentinsurface of the control group without any treatment (FIG. 13A to FIG.13D). On the other hand, in the experimental group treated with thepeptide, it can be seen that most of the exposed dentinal tubules wereclosed (FIG. 13E to FIG. 13H).

This study was supported by the Korea Evaluation Institute of IndustrialTechnology (KEIT) and funded by the Ministry of Trade, Industry & Energyin 2017 (10078369, “Development of desensitizer using functional peptideinducing dentin regeneration”).

While one or more embodiments of the present invention have beendescribed with reference to the figures, it will be understood by thoseof ordinary skill in the art that various changes in form and detailsmay be made therein without departing from the spirit and scope of thepresent invention.

1. A peptide consists of any one amino acid sequence of SEQ ID NOS: 1 to128.
 2. The peptide of claim 1, wherein the peptide consists of any oneamino acid sequence of SEQ ID NOS: 1 to
 24. 3. The peptide of claim 1,wherein the peptide consists of any one amino acid sequence of SEQ IDNOS: 25 to
 48. 4. The peptide of claim 1, wherein the peptide consistsof any one amino acid sequence of SEQ ID NOS: 49 to
 128. 5. The peptideof claim 1, wherein the peptide is for promoting regeneration of hardtissue, and treating a dentin-dental pulp disease or periodontaldisease.
 6. The peptide of claim 1, wherein the peptide has amodification selected from the group consisting of an N- or C-terminalacetylation, amidation, or methylation; a D-amino acid introduction; apeptide bond modification selected from the group consisting of CH₂—NH,CH₂—S, CH₂—S=0, and CH₂—CH_(2;) a backbone modification; and aside-chain modification.
 7. A polynucleotide encoding the peptide ofclaim
 1. 8. An expression vector comprising the polynucleotide of claim7.
 9. A method for promoting regeneration of hard tissue in a subject inneed thereof, comprising administering the peptide of claim 1 to thesubject.
 10. A method for treating dental-dentin pulp disease orperiodontal disease in a subject in need thereof, comprisingadministering the peptide of claim 1 to the subject.
 11. A compositioncomprising the peptide of claim
 1. 12. The composition of claim 11,which is a pharmaceutical composition, a dietary supplement, or afoodstuff